Two novel mutations within FREM1 gene in patients with bifid nose

Bifid nose is a rare congenital anomaly with unclear underlying etiology. Patients with Tessier No.0 and 1 type craniofacial cleft usually presented obvious bifid nose [1]. Clinical presentations vary widely from a simple groove at the nasal tip to a maxillary cleft. Nonetheless, few cases have been reported because of its rarity. Neither optimum time for surgery nor universal agreement about a certain management has been established. Therefore, instructive diagnosis and treatment needs to be established.

The developmental origin of bifid nose has not been clearly clarified, as the vertebrate face development is remarkably intricate and dynamic [2]. Understanding early nasal development stages may aid in acquainting why certain phenotypes occur. There are three major tissue blocks in the mid and upper face: the frontonasal process (FNP), lateral nasal structures and the paired maxillary processes [2]. FNP fused with the maxillary primordia formats the midline tissue such as nasal bridge and nasal tip, which dates to the 4^th week of gestation. The paired maxillary processes form the upper jaw, cheek bones and lateral nasal structures. The FNP and maxillary processes are composed of migratory neural crest [3]. The growth and maturation procedure are orchestrated by complex tissue interactions, genes network and regulatory molecules. Bifid nose occurs when the midline two nasal processes are failure to fuse. Multiple signaling pathways such as FGF, Wnt, ZIC2, PAX3, BMP, TFAP2α, DLX5 and MSX1/2 regulate neural crest cells development [4]. It’s still unclear whether the malunion is caused by the alteration in the epithelial-mesenchymal interactions or other factors including chemicals, oligohydramnios, maternal metabolic imbalances, radiations and infection [1, 5].

The FRAS1-related extracellular matrix 1 (FREM1) is located at human chromosome 9p22.3 [6]. It is widely expressed in the developing embryo in regions of epithelial/mesenchymal interaction and epidermal remodeling, which can potentially affect the craniofacial and renal development [7]. FREM1 protein belongs to the FRAS1/FREM family of extracellular matrix proteins which localizes at the basement membranes and forms a ternary complex including FRAS1, FREM1 and FREM2. Recessive mutations in FREM1 have been described to cause congenital diaphragmatic hernia and two rare syndromes—bifid nose with or without anorectal and renal anomalies syndrome (BNAR; OMIM #608,980) and Manitoba oculotrichoanal syndrome (MOTA; OMIM #248,450)—whose phenotypic characteristics overlap those seen in individuals with Fraser syndrome [8]. Therefore, the mutations in FREM1 may correlate with bifid nose, and the specific mechanisms involved still need to be further studied.

In the present study, we detected new mutation sites of FREM1 by whole exome sequencing and first-generation sequencing, which have not been reported in previous studies. The results can broaden the mutational spectrum of FREM1 in bifid nose. Moreover, the use of simple Z-plasty surgery can be well used for correction of nasal deformity in patients with mild cleft nose. This surgical management was based on our experience of more than 10 years in the treatment of congenital craniomaxillofacial malformations.

The follow-up time ranges from 2 to 3 years (2.4 ± 1.2 years), and there are 23 patients in total. The age of them ranges from 2 to 12 years old (average 4.8 years old). During the early postoperative stage, there was mild ruddy incision during the first three months. One patient had a mild infection after surgery, which was treated by partial iodophor disinfection and oral antibiotics for 3 days. Healing procedure was uneventful. There were no complications of flap ischemia, necrosis and poor healing. The wound healed primarily within about 2 weeks and the scars became invisible 3–6 months after surgery. The parents of patients were much satisfied with the aesthetical outcome (94.3%), incision scar (97.1%) and continuity of nose tip curvature (97.1%). The average very and mostly satisfactory percentage is 96.2% (Table 2).

Bifid nose, also referred to as a double or cleft nose, manifests in a diverse array of phenotypes and clinical entities. It results from abnormal embryological development of the nose. In 1976, Tessier observed more than 300 cases basing on his experiences and established the craniofacial clefts classification. The clefts were classified into 0 to 14 types according to their relationship to the zero line [10]. Middle bifid nose was commonly classified as Tessier No.0 craniofacial cleft [1]. Most cases are sporadic. Clinical presentations are complex and appear different degrees of severity. The nasal septum can be duplicated, thick or absent, and alar cartilages can be separated and nasal tip may be faintly or deeply furrowed [1]. Many other anomalies can be associated with bifid nose, such as cleft lip, orbital hypertelorism and even deformity of other systems like genitourinary [2].

Genomic technology advent has aided in profound change in many aspects, especially for rare genetic disorders. Bifid nose often overlaps with other complex syndromes, and molecular testing is critical. Gene identification promotes molecular diagnosis and gene identification. Multiple genetic mutations have been reported to be associated with bifid nose. Anyane-Yeboa et al. reported five bifid nose individuals of a family and proposed it was likely an autosomal dominant trait in 1984 [11]. Toriello et al. made a similar point [12]. With the advancement of detection technology and the reporting of more cases, many genetic mutations have been reported to be associated with the occurrence of bifid nose. Pai syndrome can represent bifid nose and a de novo apparently balanced reciprocal translocation, 46,X,t(X;16) was described [13]. Gene mutation in EFNB1 can result in bifid nose, such as c.373G > A [14], c.270_271del [15] and c.451G > A [16]. ZIC2(c.1599 * 954 T > A) [17]、PORCN(c.727C > T) [18]、TBX1(c.1132G > A) [19] have also been reported to present bifid nose. Frontonasal dysplasia resulted from ALX1, ALX3, ALX4 can also present bifid nose [2]. There have been many case reports of MOTA syndrome and BNAR syndrome, which are also related to the FREM1 gene. The patients with MOTA syndrome may present a broad or bifid nasal tip, cryptophthalmos, microphthalmia, eyelid colobomas, an aberrant hairline, and gastrointestinal anomalies such as omphalocele and anal stenosis [6].

Herein we report two novel mutations in FREM1 gene: heterozygous frameshift mutation c.870_876del and heterozygous missense variation c.2 T > C. They have not been reported previously. The novel frameshift variant c.870_876del causes premature termination codon and variant c.2 T > C causes the start lost. As a quality control pathway, nonsense-mediated decay may remove the premature termination codons, which is a possible alternative pathogenic mechanism [20]. Only a dozen different FREM1 mutations have been reported, and few animal models have been described [21]. FREM1 protein concludes 12 chondroitin sulfate proteoglycan (CSPG) repeats, a putative signal sequence, a calx-β domain and a C-terminal type C lectin-like domain [7]. FREM1 is widely expressed in some neural crest mesenchyme, it can be found in many syndromes such as Bifid Nose Renal Agenesis and Anorectal malformations (BNAR) and Manitoba-oculo-tricho-anal (MOTA) [22]. The interaction between different cell types and the availability of different extracellular ligands for the cognate receptors are thought to have the participation of FREM1 [23]. FRAS1, FREM1 and FREM2 gene have been shown to encode a group of extracellular matrix proteins, forming a ternary complex which locates at the basement membrane [24]. Therefore, the correct expression of FREM1 is necessary for the normal development of nasal morphology. The interactions will be disrupted if there are loss-of-function FREM1 mutations. There have been several reported variations, such as loss of the exons 19 to 30 [7], exon 8–23 deletion [6], heterozygous c.3939 A > C (p.Y1313X) variant at exon 23 and heterozygous c.580G > A (p.R194X) variant at exon5 [25]. The animal models of related research have also been mature [6, 24, 26‐28]. However, the relationship between the specific mutation site and the phenotype has not established, which needs continuing to be explored. Chacon-Camacho et al. [21] summarized 27 patients with FREM1 mutations, we sorted other reported patients in Table 4. We found that the patients with mutations in FREM1 generally had changes in nasal morphology, but the symptoms in other areas vary. Gender is also an influencing factor [7], we hypothesize that this is why this pair of twins has bifid nose and no other symptoms. This article further supplements the understanding of bifid nose-related genes.

As the bifid nose is the most common craniofacial cleft, many surgical techniques have been proposed basing on personal experience and preference. However, no surgical technique has been universally accepted. The surgical treatment still present great challenge due to limited number of publications and complexity of malformation. Nasal deformities correction concludes skeletal and soft tissue malformation. ROE first proposed public correction of bifid nose in 1887, and a second stage completion surgery was first advised by Kazanjian and Holmes [1]. Kurokawa performed dermal graft via the nasal dorsum and applied on the nasal apex [35]. Ali Tawfik combined Millard forked flap with external rhinoplasty and successfully helped six patients. It increased the scar and secondary operation was usually needed [1]. Tuersunjiang et al. made an inverted-V transcolumellar incision, modified the shape of nose and achieved good results [36]. Rib cartilage has inherent structural advantage. Many surgeons recommend rib cartilage as the best autologous material in rhinoplasty. In 1917, Selfridge first emphasized rib cartilage as nasal reconstruction graft material [37]. Recently more and more nasal reconstruction via rib cartilage have been reported [38]. Researchers used autologous bone tissue or cartilage to treat deformity and got good functional and aesthetic outcomes [39, 40]. However, some surgeons avoid using cartilage [41]. First, 2 anatomic sites prolong the time of operation and staying in the hospital, which not only results in a late discharge but also increases the hospital expenses. Some patients can’t afford it. Second, chest tube insertion and pneumothorax might occur during operation. The occurrence of these unexpected situations can bring other troubles. Third, the cartilage may twist or bend postoperatively. If so then the secondary operation is needed and patients will express dissatisfaction. Fourth, cartilage may occur calcification. Notably, this method is not suitable for children because they are growing and developing. The operation will affect their cartilage development. What’s more, these patients with mild bifid nose don’t need to raise their nose. Overall, diverse surgical methods have been proposed but they each have their pros and cons. There is still no widely accepted approach. Although there have been some studies to use local flaps, surgical treatment for mild bifid nose is rarely reported. We aim to identify a technique to achieve more permanent and effective correction of mild bifid nose. The Z-shaped incision could be an ideal option to help patients who just have a small groove at the tip of nose. The results were stable and pleasant. Most importantly, the skin is much coherent in this method and excess skin doesn’t need to be excised. The excess skin facilitates later implantation of cartilage or prosthesis when the child is older. Otherwise the later improvement is limited. This technique described in this article can be applied to all the similar patients. We have improved the aesthetics as much as possible with the smallest scar.

The optimum age of plastic surgery for bifid nose is an arguable issue. Some surgeons recommend not to operate on the pediatric nose because there is potential damage to the nasal growth. Doval et al. [32] retrospectively found that surgeries performed on child still have a good effect. We prefer the patients being operated at the age of between 3 and 6 years old. The anesthesia of children who are too small may affect the nervous system of children, and children will have a stronger self-awareness of appearance after the age of 6. Otherwise the patients may suffer teasing while dealing with their mates.

The surgical treatment proposed in this article can address problems such as chromatic aberration and low survival rate, which often occur in local skin flaps. The procedure is simple but can effectively improve the patients’ appearance. No serious surgical complications have been found so far except mild infection or edema. The limitations of this approach include the number of patients is relatively small and the follow-up time needs extension. The genetic test included only one set of twins and their parents, and samples from more patients could provide richer results. Most importantly, the surgical method presented is only suitable for simple short and wide nose tip, it is not suitable for more serious ones.

Our discoveries enrich the understanding of bifid nose and broaden the mutational spectrum, which enables more patients to receive personalized treatments. It’s still needed to be open to unexpected scenarios such as richer genetics understanding and better surgical methods, in the continuing way for the bifid nose.